Project Summary Unconventional signaling by the R-spondin family of WNT regulators The four R-spondins (RSPOs1-4) are vertebrate-specific secreted proteins that dramatically amplify signaling through the WNT/?-catenin pathway, a cell-cell communication system that regulates tissue patterning during development and regenerative responses in adults. Mutations that damage the RSPO signaling circuit cause structural birth defects, exemplified by limb truncation, lung hypoplasia and craniofacial malformations seen in patients with mutations in RSPO2. In adults, RSPOs function as niche-derived signals required for the renewal of epithelial stem cells in multiple tissues, including the intestine, skin, and bone. RSPOs amplify WNT signals by simultaneously binding to two receptors: Leucine-rich repeat-containing G-protein coupled receptors (LGRs) and the ZNRF3/RNF43 transmembrane E3 ubiquitin ligases. RSPOs increase WNT receptor levels only in specific cell types, such as intestinal stem cells, that express LGR receptors and thus allow the strength of the WNT signal (a potentially oncogenic signal) to be tightly controlled in time and space. While LGRs had been considered obligate high-affinity receptors for RSPOs, we made the unexpected discovery (along with other groups) that RSPOs can amplify WNT signals in the absence of all LGRs (?LGR-independent signaling?) or in the absence of ZNRF3/RNF43 (?ZNRF3/RNF43-independent signaling?). Distinct from the intensively studied LGR-mediated signaling in intestinal stem cells, LGR-independent signaling is particularly relevant for RSPO- related birth defects: it is the dominant mode of signaling during development of the limbs, lungs and cardiovascular system. Our preliminary results show that alternate, undiscovered receptors must exist that mediate some cellular responses to RSPOs. To identify these receptors and associated signaling components, we propose an innovative strategy that combines ligand engineering with genome-wide loss-of-function screens in cultured cells. In Aim 1, we use chimeric and mutant RSPO ligands in cell and organoid culture systems to test the hypothesis that Heparan Sulfate Proteoglycans (HSPGs) can function as RSPO co-receptors to transduce LGR-independent signals. In Aim 2, we use engineered RSPO ligands constrained to signal through either LGR-independent or ZNRF3/RNF43-independent modes to search for the required genes using CRISPR- based screens and haploid genetic screens. Successful completion of this project will identify the signaling machinery that mediates cellular responses to RSPOs in developmental and regenerative contexts, thereby improving our understanding of WNT-related structural birth defects and providing new strategies to enhance WNT-regulated regenerative responses in a tissue-selective manner.